Biochemistry/Molecular Biology - ARVO

ARVO 2013 Annual Meeting Abstracts by Scientific Section/Group - Biochemistry/Molecular BiologyMethods: We performed morpholino experiments in zebra fish totest the functional relevance of Reep6 in retina development. Tostudy the transcriptional regulation of Reep6 gene, we used acombined approach including rapid analysis of cDNA ends (5’-RACE), chromatin immunoprecipitation, luciferase reporter assay,promoter mapping in retina explant and in vivo shRNA analysis.Results: Knockdown of Reep6 in zebrafish by morpholinos led tomicrophthalmia. NRL binding to the Reep6 intron enhanced itspromoter activity in a luciferase reporter assay and also in a GFPreporter assay in retina explant.Conclusions: Reep6 expression is controlled by NRL through anenhancer located within its intron. NRL direct target genes serve asexcellent candidates for retinal disease gene discovery.Commercial Relationships: Hong Hao, None; Shobi Veleri, None;Bo Sun, None; Raman Sood, None; Paul Liu, None; AnandSwaroop, NoneSupport: NEI intramural supportProgram Number: 2021 Poster Board Number: D0052Presentation Time: 11:00 AM - 12:45 PMElucidating the mechanism behind enhanced retinal transductionof an AAV2 variantKenton T. Woodard, Richard Samulski. Gene Therapy, Neurobiology,Univ of NC at Chapel Hill, Chapel Hill, NC.Purpose: Photoreceptor dystrophies lead to reduced vision as thephotoreceptors degenerate. Gene therapy using Adeno-associatedvirus (AAV) has shown to be safe and effective at preservingphotoreceptors. This preservation is robust when AAV is deliveredsubretinally, but inefficient when delivered intravitreally (IVit) eventhough IVit delivery of AAV offers practical advantages. Of thenaturally occurring AAV serotypes, most don't transduce the retinaIVit, and of the serotypes that can, transduction is limited to theganglion cell layer. Efforts are now underway to develop mutantAAVs that are capable of photoreceptor transduction via IVitdelivery.Methods: We developed mutations on the AAV2 capsid whichdisplayed enhanced transduction of multiple retinal cell layers acrossseveral animal models. This mutant was dubbed AAV2.5. Ourinterest was to determine the mechanism of action for thisenhancement. We assessed difference in receptor usage and receptorbinding affinity of AAV2 and AAV2.5 in vitro using several celllines and affinity chromatography. To identify key amino acidsinvolved in the enhancement, we generated chimeric capsid carryingeach of the 5 amino acids modified in AAV2.5 separately andproduced viral preps carrying reporter gene for testing in vivo.Finally, we tried to promote greater transduction in the retina bymixing soluble heparin with AAV2 and AAV2.5.Results: We saw no difference in receptor usage or affinity betweenAAV2 and AAV2.5, indicating a still unknown mechanism for theenhancement in the retina. Similar to published results in muscle, thethreonine insertion found in AA2.5 but absent from AAV2 seemed tobe a key amino acid necessary for increased retinal transduction.While soluble heparin mixed with AAV2 showed a greater number ofretinal cells being transduced, soluble heparin mixed with AAV2.5showed no increase in transduction.Conclusions: The lack of difference in primary heparin sulfatereceptor usage and affinity may indicate that AAV2.5 has asecondary receptor usage or downstream advantages in the viralendocytic pathway. Current work is aimed at determining whatmechanisms are at play for enhanced retinal transduction, notably byreal time imaging of capsid particle critical steps. Future work willinvestigate newly identified amino acid insertions in combinationwith chimeric AAV2.5 to fully exploit maximum robust transductionprofile in ocular target tissue.Commercial Relationships: Kenton T. Woodard, None; RichardSamulski, Asklepios BioPharmaceutical, Inc. (I)Support: NEI Grant EY0199555-01Program Number: 2022 Poster Board Number: D0053Presentation Time: 11:00 AM - 12:45 PMCharacterization of Age Related Maculopathy Susceptibility 2(ARMS2) transcripts in human and Cynomolgus macaque retinaPeng Yu, Darius Donohue, Kristina L. Rhoades, Carl Romano,Rajkumar V. Patil. Opthalmology, Novartis Institutes for BiomedicalResearch, Alcon Research Ltd., Fort Worth, TX.Purpose: ARMS2 gene is the second major risk allele for AMD,contributing independently of complement factor H to disease risk.The rs10490924 (A69S) SNP in ARMS2 accounts for approximately35% of the population-attributable risk for the development of AMD.However, there are controversies regarding its function, localization,expression and whether it is a real gene or pseudo gene. The presentstudy was undertaken to gain an understanding of ARMS2 transcriptsin human and macaque retina.Methods: Total RNA from human and macaque retinas was reversetranscribed and PCR and qPCR were performed using customdesigned primers and probes. For macaque, primers were designedbased on homologous human sequence. The cDNAs of varioushuman tissues including placenta were purchased from Zyagen. ThePCR products of interest were purified and sequenced to confirm theauthenticity of the amplified products.Results: PCR and qPCR results showed that two splice variants ofARMS2 mRNAs are expressed in human retina. DNA sequencingresults of PCR products revealed that a larger splice variant ofARMS2 (designated as variant A) was detected in 5 human retinaswith more common alleles at R38 and A69, whereas, a shorter splicevariant of ARMS2 (designated as variant B) was detected in 7 humanretinas and all of them showed “C to T” SNP at rs2736911.Quantitative RT-PCR results suggested that expression level ofvariant B was much higher than the variant A (the Δcycle is about 4-5). In macaque retina, only variant A was detected. In human,ARMS2 expression was detected only in placenta besides retina.Conclusions: Two ARMS2 splice variants are expressed in humanretina, whereas, only variant A is expressed in macaque retina.Variant B is the major transcript of ARMS2 in human retina. Ourresults suggest that variant A carried more common alleles at both,R38 and A69 positions, whereas, variant B always carried R38X SNPand common allele at A69 suggesting that R38X variation may be aprerequisite for variant B splicing. Further studies are required tounderstand the function, cellular localization, and the pathogenic roleof ARMS2 splice variants in the development of AMD.Commercial Relationships: Peng Yu, Alcon/Novartis (E); DariusDonohue, Alcon/Novartis (E); Kristina L. Rhoades, Alcon/Novartis(E); Carl Romano, Alcon/Novartis (E); Rajkumar V. Patil,Alcon/Novartis (E)Program Number: 2023 Poster Board Number: D0054Presentation Time: 11:00 AM - 12:45 PMGeneration of GARP2-Specific Knockout Mice Using Zinc FingerNuclease TechnologySteven J. Pittler 1 , Delores Davis 1 , Larry W. Johnson 2 , Robert A.Kesterson 2 . 1 Vision Sciences, Univ of Alabama at Birmingham,Birmingham, AL; 2 Genetics Research Division, Univ of Alabama atBirmingham, Birmingham, AL.Purpose: The Cngb1 locus encodes the rod photoreceptor cGMPgatedcation channel β-subunit and two soluble glutamic acid rich©2013, Copyright by the Association for Research in Vision and Ophthalmology, Inc., all rights reserved. Go to iovs.org to access the version of record. For permissionto reproduce any abstract, contact the ARVO Office at arvo@arvo.org.